臺灣博碩士論文加值系統

近年來，藉助螢光顯微鏡的發展與螢光蛋白質技術的改良，開始利用螢光技術觀察生物分子在原核生物中即時的分佈及作用情形，發現細胞質內有許多與真核細胞相似的系統，如細胞骨架系統（Cytoskeleton）、訊號傳遞系統等，進一步發現許多蛋白質在細胞內有其特殊的位置分佈（Localization），以執行相關的生命功能。本篇研究目標就是探討端鞭毛形成初期的組成蛋白FliG，探討FliG在細胞不同生長時期在細胞內的分佈（Localization）行為（Behavior）。另外，前人研究指出受到一些環境壓力如飢餓、活而不長狀態（VBNC），或化學物處理如A22等，腸炎弧菌會從桿狀變成圓形，因此我們也想瞭解在環境壓力處理形成的圓形或回復後的桿狀細胞在轉換期中，FliG在細胞中的行為表現，及FliG表現與實際細胞鞭毛形成的相關性。我們以綠色螢光蛋白（GFP）與我們的目標蛋白FliG做N端連結，建立具有強啟動子（PA1/04/03）的低拷貝數質體（Low copy numbers plasmid），做成pSC31，放入腸炎弧菌中表現，以螢光顯微鏡來觀察FliG的行為。本篇研究發現FliG在細胞中會隨著不同的細胞時期或壓力產生聚集、分散、甚至是移動的現象。我們同時以RT-PCR來測定fliG及其他鞭毛構成蛋白質，fliF（Basal body）、flgE (Hook)、flaA (Flagellin)等在細胞週期及不同環境壓力下及回復時的基因表現。配合上電子顯微鏡的結果，我們發現野生株VP1137（Wild type）在Log phase與Stationary phase都有鞭毛的形成，在培養三天的Prolong stationary phase有兩種不同形式的菌體，一為小而黑色不具鞭毛的菌體，另一則是膨大而具有鞭毛的菌體，並且在膨大菌體上有許多類似囊泡（Vesicle）的突起。在飢餓的條件下，菌體逐漸接近球形，直到處理三天後的樣本依然具有鞭毛，RT-PCR結果亦支持顯微鏡的觀察。在生長對數期觀察轉殖株VP1137sc31的GFP-FliG訊號時發現其具有移動且有時會有兩個聚集的現象，在電子顯微鏡的觀察上我們發現加入質體的菌體中有一個到兩個端鞭毛。我們在RT-PCR結果顯示fliG與fliF會有輕微過量表現（Mild overexpression）的現象。不論是VP1137或是VP1137sc31，其鞭毛的形成位置似乎都會隨著細胞週期改變位置，除了大部分會出現在端點外，也有一部份會出現在1/4、1/2的位置，但是出現時大部分都是在細胞分裂的過程前期，最後會形成兩端都具有鞭毛的細胞，這與我們在GFP-FliG上的觀察相似。GFP-FliG在細胞中為自我聚集(Self-assembly)成鞭毛的樞紐(Switch) ，研究中顯示GFP-FliG聚集平時穩定的存在於細胞端點，但在細胞分裂時往細胞中間移動，在新GFP-FliG聚集形成後，又回到端點。MreB在細菌中決定很多蛋白質的特殊位置，我們使用MreB去聚集化（depolymerize）的試劑A22，處理VP1137sc31發現並不會影響到GFP-FliG spot的端性（polarity），顯示MreB 並不參與決定GFP-FliG spot的端性分佈。穿透式電子顯微鏡的結果也顯示細菌的鞭毛會有移動的現象，但是如何移動，及新的鞭毛何時形成，皆有待進一步探討。

Prokaryotic cells are similar to eukaryotic cells and have same mechanisms or molecular behaviors in cytoplasm. The most frequently studied topics are cytoskeletons and pathogenesis related proteins in prokaryotic cell. There are distinguished polarity marker proteins of polar flagella or pili, but relative few researches have been reported. In this study, we aimed on FliG, the early stage protein of polar flagellum formation, as a localization marker. FliG is switch complex of the basal body of polar flagellum in Vibrio parahaemolyticus and plays a key function for the polar flagellum. It accepts signals or energy from chemotaxis signaling protein, fixes basal body with stator protein, and becomes a rotor protein at the same time. The polar flagellum proteins could target to cell pole. FliG in a polar flagellum is then a distinct marker of cell polarity. FliG is secondary stage protein of polar flagellum assembly. The self-assembly mechanism of switch complex (FliG) and its duplication is unclear. We constructed a functional GFP-FliG fusion protein and visualized the behavior of FliG in time course or time lapse ways by fluorescence microscopy. We induced the rod shape cell to become spherical and then recovered, and the behavior of GFP-FliG was observed. GFP-FliG cluster was movable in cells in the division stage, and assembled and disassembled during different stages of the life cycle or under stresses. GFP-FliG showed characteristic polar localization regardless about the change of cell shape, unless the cell was in the division stage. When cells were treated under stress for a long period, the GFP-FliG cluster disassembled. Cells grown in rich medium, GFP-FliG showed smear expression and then spot formed in the centric or eccentric positions, and eventually moved to the pole. We treated cells with A22, an inhibitor of MreB and examined the changes of GFP-FliG. The treated cells became spherical shape while the polar localization of the GFP-FliG clusters was not disturbed. The results showed that MreB was not associated in the movement and localization of GFP-FliG. Reverse transcription- Polymerase Chain Reaction and electron microscopy experiments revealed that FliG behavior was closely related to the expression of polar flagellum in different growth phases and in starvation state. Cells with GFP-FliG gene (VP1137sc31), showed one or two GFP-GFP clusters and same numbers of polar flagellum in exponential phase as observed by TEM. Reverse transcription- Polymerase Chain Reaction revealed that gfp-fliG and gfp-fliF exhibited mild over expression as compared to fliG and fliF. In wild type cells, the polar flagella were not always in the polar position. Some of these flagella shifted to other positions in early stage during cell division, and then two polar flagella were found in opposite poles in late stage. The mechanism of the formation and movement of FliG cluster and polar flagellum need further exploration.

TABLE OF CONTENTS


TABLE OF CONTENTS I
LIST OF TABLES IV
LIST OF FIGURES V
LIST OF SUPPLEMENTARY DATA VIII
中文摘要 1
ABSTRACT 3
INTRODUCTION 5
I. Polarity in bacteria 6
Oscillation causes bipolar localization 8
Polarity determinates by protease 8
Preposition hypothesis 9
Birth scar 9
A. MreB, a determinant of protein localization 10
B. Flagellum localization in Caulobacter crescentus 11
II. Stress adaptation 13
A. Shape determinate 13
B. Stresses influence cell shape 15
Sporulation 15
Filamentations 15
Miniaturization 16
III. Studies on Flagella 18
A. Polar Flagella 19
Flagellar filament 19
Hook 20
Sheath 20
Basal body 21
The Switch 22
B. Chemotaxis system and flagellar basal body 22
C. FlhF-FlhG pair adjusts polar flagellum formation 24
IV. Vibrio parahaemolyticus 26
A. Basic characteristics of Vibrio parahaemolyticus 26
B. Polar flagellum in Vibrio parahaemolyticus 27
OBJECTIVE OF STUDY 29
MATERIALS AND METHODS 30
Bacterial strains and growth conditions 30
Plasmid construction 31
Transformation of pSC31 into E. coli XL1-Blue 32
Transformation of pSC31 into E. coli s17 λpir by electroporation 33
ECSsc31 conjugated to V. parahaemolyticus 1137 33
Preparation of cells for microscopy 34
Image Acquisition and Processing 35
Time-Lapse Imaging 36
Polar flagellum staining by Alexa Fluor 532 36
Transmission electron microscopy 37
A22 treatment 38
Cell membrane stain 38
VAN-FL staining 38
Reverse transcription-polymerase chain reaction (RT-PCR) 39
RNA purification 39
Elimination of contaminating DNA 40
RNA quantitative determination 40
Reverse transcription 40
PCR procedure 41
RESULT 42
Basic study on GFP-FliG contained strain 42
Behavior of GFP-FliG 46
Cell cycle 46
GFP-FliG behavior in cells under stress 46
GFP-FliG behavior in cells recovered from stress 47
Factors controlling the FliG assembly 48
DISCUSSION 50
GFP-FliG roles in Vibrio parahaemolyticus 1137sc31 50
GFP-FliG behavior in Vibrio parahaemolyticus 1137sc31 51
Probable mechanism of switch complex formation in Vibrio parahaemolyticus 53
MS ring, switch complex, and C ring interaction 54
FliG polarity was not determined by MreB 56
Conclusion 57
REFERENCE 58




LIST OF TABLES


Table1. Bacterial strains used in this study. 71
Table 2. Plasmids used in this study. 72
Table 3. Primers used in this study. 73
Table 4. Cell morphology and fluorescence pattern of Vibrio parahaemolyticus 1137sc31 in nutrition broth. 74
Table 5. Cell morphology and fluorescence pattern of Vibrio parahaemolyticus 1137sc31 starved in MMS- 0.5% NaCl for different intervals. 75
Table 6. Cell morphology and fluorescence pattern of Vibrio parahaemolyticus 1137sc31 induced into VBNC state. 76













LIST OF FIGURES


Fig. 1 Polar flagellum patterns in Vibrio parahaemolyticus 1137 observed by TEM examination. 77
Fig. 2 Relative expression levels of fliG, fliF, flgE and flaA genes in Vibrio parahaemolyticus 1137 detected by RT-PCR. 78
Fig. 3 fliG expression was determined by RT-PCR in Vibrio parahaemolyticus 1137 under different treatments. 79
Fig. 4 fliF expression was determined by RT-PCR in Vibrio parahaemolyticus under different treatments. 80
Fig. 5 flgE expression was determined by RT-PCR in Vibrio parahaemolyticus under different treatments. 81
Fig.6 flaA expression was determined by RT-PCR in Vibrio parahaemolyticus under different treatments. 82
Fig. 7 Polar flagellum pattern observed in Vibrio parahaemolyticus 1137 by TEM. 83
Fig. 8 pSC31 construction map. 84
Fig. 9 GFP-FliG 3D structure analysis 85
Fig. 10 fliG expression level was determined by RT-PCR in Vibrio parahaemolyticus under different treatments. 86
Fig. 11 fliF expression level was determined by RT-PCR in Vibrio parahaemolyticus under different treatments. 87
Fig. 12 Polar flagellum pattern was observed in Vibrio parahaemolyticus 1137sc31 by TEM examination. 88
Fig. 13 (A) Flagellum stain of Vibrio parahaemolyticus 1137sc31 by Alexa flour 532. (B) GFP-FliG localization (log phase) of Vibrio parahaemolyticus 1137sc31. 89
Fig. 14 Growth curve of Vibrio parahaemolyticus 1137 (○) and VP1137sc31 (●) in LB-3% NaCl, and VP1137sc31 added Kanamycin 300-μg/ml. 90
Fig. 15 Behavior of GFP-FliG during cell division in Vibrio parahaemolyticus 1137sc31. 91
Fig. 16 Time course of GFP-FliG in Vibrio parahaemolyticus 1137sc31 cultured in LB-3% NaCl and Kanamycin 300-μg/ml. 92
Fig. 17 Movement of GFP-FliG spots in Vibrio parahaemolyticus 1137sc31in different stages of the cell cycle. 93
Fig. 18 Localization of GFP-FliG in starved Vibrio parahaemolyticus 1137sc31 cells. 94
Fig. 19 Induction of Vibrio parahaemolyticus 1137(○) and Vibrio parahaemolyticus 1137sc31 (●) induced into VBNC state. 95
Fig. 20 GFP-FliG spot pattern change of Vibrio parahaemolyticus 1137sc31 cell in VBNC state. 96
Fig. 21-1 One spot GFP-FliG patterns at cell shape transition in Vibrio parahaemolyticus 1137sc31. 97
Fig. 21-2 Two spots GFP-FliG patterns at cell shape transition in Vibrio parahaemolyticus 1137sc31. 98
Fig. 22 GFP-FliG behavior was observed during the recovery of stationary phase spherical cells of Vibrio parahaemolyticus 1137sc31. 99
Fig. 23 Association of the GFP-FliG was located with the change of cell shape in Vibrio parahaemolyticus 1137sc31. 100
Fig. 24 GFP-FliG spot pattern was related to new growth direction in Vibrio parahaemolyticus 1137sc31. 101
Fig. 25-1 GFP-FliG localized in Vibrio parahaemolyticus 1137sc31 cells treated by different level of A22 for different intervals. 102
Fig. 25-2 GFP-FliG localized in Vibrio parahaemolyticus 1137sc31 cells treated by different level of A22 for different intervals. 103
Fig. 25-3 GFP-FliG localized observed in Vibrio parahaemolyticus 1137sc31 cells treated with A22 10-μg/ml at 37℃ for 1h. 104
Fig. 26 Van-FL stained Bacillus subtilis BCRC10255 and Vibrio parahaemolyticus 1137 by published method showing the new born cell wall patterns(Daniel and Errington, 2003). 105
Fig. 27 Van-FL stained Vibrio parahaemolyticus 1137 by our modified method to show the new born cell wall patterns. 106
Fig. 28 Van-FL stained Vibrio parahaemolyticus 1137 by our modified method to show the new born cell wall patterns of the VBNC recovered cells. 107













LIST OF SUPPLEMENTARY DATA

Supplementary Table 1. Examples of bacterial proteins that localize to the cell pole (Lybarger and Maddock, 2001). 108
Supplementary Figure 1. Graphical Overview of the Model for TipN and TipF Function at the Division Plane and the Newborn Pole. 109
Supplementary Figure 2. Morphogenetic pathway for the flagellum of Salmonella. 110
Supplementary Figure 3. YFP-MreB localization treated with A22. 111

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